Protein blot (Western)

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Protein gel (1st step of a Western blot) showing a prestained ladder on both sides and Coomassie-stained protein samples in the centre. PS: You wouldn't stain with Coomassie before labelling with antibodies. This is for better illustration.

A Western blot, also known as the immunoblot or protein blot, is a method of semiquantitative determination of protein expression. Crude cell lysates are loaded into a polyacrylamide gel containing a denaturing agent which give all the proteins a net negative charge. A current passing through the gel will then propel the proteins through the gel, with the largest proteins travelling the slowest. This results in the proteins being roughly separated by their mass. The proteins are then transferred from the gel onto a membrane (often nitrocellulose or PVDF), which is then incubated with an antibody directed against the protein of interest. By using a detector conjugated to an antibody one can then specifically detect the protein of interest.

Standard western blot procedure

Prior to running the western blotting experiment, consult with the vendor in order to determine if they have optimized the titration for the antibody you just purchased. Most vendors should offer some guideline for how to use the antibody. Value your time and effort first and foremost. If the experiment comes out strange, make the call or email the vendor for assistance. Your time is the most valuable element to the procedure!

II) Transfer proteins to a nitrocellulose or PVDF membrane and block in fresh 5% milk TTBS 2 hr, r.t. or overnight at 4C. Perform 3 shake rinses and drain until no residual milk appears to stream off the wash buffer.

Make sure to wash between steps with ambient or cold TTBS at least 4X for 5 minutes each wash.

Perform ECL using a suitable detection reagent.

Optimize protein signal with multiple exposure times.

Phosphorylation state specific western blot procedure

Adjusting certain incubation conditions can improve the detection signal for the phospho-specific antibody. When serine/threonine phosphatase inhibitor Sodium Fluoride (NaF), and tyrosine phosphatase inhibitor Sodium Orthovanadate (Na3VO4) are included in the blocking and incubation buffers, phospho-specific signals can noticeably improve. Including 50 mM NaF and 5 mM Na3VO4 in the blocking and incubation buffers can improve the signal. Using 5% milk diluent for primary and secondary incubations will reduce the nonspecific banding and background.

Primary antibody: Use a diluent containing a blocking protein (5% milk TTBS or 1%milk/1%BSA TTBS) to dilute the primary antibody.

Secondary antibody: Use a diluent containing a blocking protein (5% milk TTBS or 1%milk/1%BSA TTBS) to dilute the primary antibody. Performing a "Secondary control" control blot where the primary antibody is purposely omitted is useful for identifying whether nonspecific signals are due to the secondary antibody conjugate or the primary antibody.

Washes: All wash steps are critical for reducing general background signal and nonspecific binding to discrete bands. If high background is a problem, the number, length and composition of the washes can all be increased.

PVDF versus Nitrocellulose

The western blotting procedures are the same for PVDF or nitrocellulose, however the handling of these membranes are different prior to- and during- transfer of proteins from the SDS-PAGE gel to the membrane.

Nitrocellulose exhibits the highest sensitivity with very low backgrounds in all transfers, especially in protein blotting. Unlike PVDF, nitrocellulose wets out naturally, does not require methanol, and will not turn hydrophobic during the transfer process. Nitrocellulose is very easily blocked and does not need the many blocking steps required with PVDF. Protocols for Western Blotting with PVDF and Nitrocellulose are the same with a few exceptions.

PVDF is hydrophobic and therefore should be prewet in methanol before it is used. Wet the membrane in methanol for 15 seconds. Membrane should uniformly change from opaque to semi-transparent. Carefully place the membrane in ultrapure water and soak for 2 minutes. Then carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes. Another change to note is that the SDS tolerances are not equivalent for PVDF and Nitrocellulose. The binding of protein to PVDF is much more sensitive to SDS levels. Too much SDS can inhibit the protein's ability to bind to the PVDF and can, in fact, help proteins already bound to the membrane to slip off. SDS levels should never exceed 0.05% for PVDF.

Membrane Stripping

Stability of reagents

Ammonium persulphate/persulfate (APS) is a very reactive compound (used for radical generation in the polymerisation process). It is prone to oxidation and loss of activity which will increase your polymerisation times. Make small aliquots, keep in the cold. [1]

TEMED, the catalyst of polymerisation, is also prone to oxidation especially if mixed with water which it attracts if the container is left open. Seal quickly, keep in cold place. [2][3]